Reprint of: Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film

Abstract

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry.

Cell culture on hydrophilicity-controlled silicon nitride surfaces

“…For example, 25-nmthick silicon nitride membranes can separate a vacuum from atmospheric pressure when the membrane size is 0.1 mm 9 0.1 mm (Nishiyama et al 2010). The membrane thickness can be controlled with nanometer-scale accuracy (Ciarlo 2002;Neumann et al 2004).…”

Platinum Blue Staining of Cells Grown in Electrospun Scaffolds

“…Recently, platinum blue has been used as a heavy metal stain for imaging cells using low-vacuum SEM (12, 14) and atmospheric SEM (15). Platinum blue is a polymeric compound of deep blue, green, or purple in which platinum is coordinated with amide groups (16).…”

Nanometer-scale Real-space Observation and Material Processing for Polymer Materials under Atmospheric Pressure: Application of Atmospheric Scanning Electron Microscopy

“…A novel electron microscopy technique using an atmospheric scanning electron microscope (ASEM) has been recently developed, 17 which enables the observation of specimens under atmospheric pressure. This new device uses as the specimen holder a 3 mL open plastic dish with a thin silicon nitride (SiN) membrane (250 µm © 250 µm, 100 nm thick) at the center.…”

“…The thin membrane separates the specimen (in the dish under atmospheric pressure) from the high-vacuum SEM optics. 17 ASEM can serve as an ideal tool for observing liquid-based specimens at nanometer-level resolution. In a previously published paper, ASEM was successfully used to observe cells in buffer solutions, 17 crystal growth processes in liquids, and the Brownian motion of silica particles.…”

“…17 ASEM can serve as an ideal tool for observing liquid-based specimens at nanometer-level resolution. In a previously published paper, ASEM was successfully used to observe cells in buffer solutions, 17 crystal growth processes in liquids, and the Brownian motion of silica particles. 18 In this study, we demonstrate example observations of polymer materials under atmospheric pressure at the nanometer scale.…”